The increasing drug resistance among pathogenic bacteria is a major problem. This project is an integrated attempt to study the two general and synergistic mechanisms that bacteria use in order to prevent the access of antibiotics and chemotherapeutic agents to their targets within the bacterial cell. Such drugs must enter the bacterium and bind to the targets in order to inhibit and kill the cell. This process is made difficult by the bacteria by first building a low permeability surface membrane. An outstanding example is the outer membrane of Pseudomonas aeruginosa or Acinetobacter spp, with their exceptionally low-permeability channels or porins. The molecular mechanisms that make the channel protein to show such a low permeability appear to be in the peculiar folding process of these porins, and the project will examine the details of this process. Another example is the mycobacterial outer membrane or cell wall with its unique lipid composition, which makes the entry of lipophilic drugs apparently quite difficult. The project will examine the lipid composition of this membrane in a quantitative manner, and the effect of the lipid composition on permeability. The second mechanism for preventing drug access is the active efflux. Thus, the bacteria can overproduce multidrug efflux pumps of surprisingly wide specificity to actively pump out drugs before they reach the target. The project will examine the AcrB pump of Escherichia coli, which pumps out most of the commonly used antibiotics, with the sole exception of aminoglycosides. It will try to elucidate the path the drug molecule follows inside the large AcrB transporter, and the details of interaction between AcrB and its accessory proteins AcrA and TolC in the adjacent compartments of periplasm and outer membrane. The effect of the presence of drugs on the assembly of AcrB with AcrA and TolC will also be examined. Many of the studies will utilize a covalently linked trimer version of AcrB, so that each component monomer can be fixed in the precise conformation representing a finite step in the transport process. It is hoped that these studies will result in a rather complete and quantitative understanding of the drug entry (and expulsion) process in gram-negative bacteria and mycobacteria, and will help in the design of better antimicrobial agents and also better inhibitors of the efflux pumps.